Dengue fever is caused by any of four serotypes of dengue virus, dengue-1, dengue-2, dengue-3, and dengue-4, which are transmitted to humans by mosquitoes. In adults, dengue infections typically cause self-limited but incapacitating acute illness with fever, muscle pains, headache and an occasional rash. The illness may be complicated by hemorrhagic fever, which may be manifested by a positive tourniquet test, spontaneous petechiae, frank bleeding, and/or shock. Dengue hemorrhagic fever is fatal in about 0.5% of cases. Patients who have antibody from an earlier dengue infection who are subsequently infected by another dengue strain have been shown to be at higher risk for dengue hemorrhagic fever.
The mosquito vectors of dengue viruses are found in all tropical and sub-tropical areas of the world and in some temperate areas of the United States, Europe, Africa, and the Middle East. In recent years, endemic and epidemic dengue infections have occured in Central and South Ameria, Southeast Asia, India, Africa, the Caribbean and Pacific regions. Vector control is impractical.
Development of live attenuated virus vaccines for dengue has been pursued by the Walter Reed Army Institute of Research (WRAIR) and Mahidol University in Thailand (Kanesa-thasan 1997), (Bhamarapravati 1997). Early efforts established that there were no known, reliable genetic or phenotypic markers or proven animal models for predicting attenuation of dengue virus. Hence, the principal means of determining the safety and immunogenicity of a candidate vaccine was to evaluate it in volunteers. Over the past forty years, many dengue vaccine strains were found to be unsuitable for humans because of over-attenuation, which rendered them insufficiently immunogenic, or because they were under-attenuated, resulting in dengue fever. Many months or years of testing were needed before a candidate vaccine could be determined to be acceptable.
A concerted investigation was undertaken at the WRAIR to select four attenuated dengue vaccine candidates, one for each serotype. As with other successful human vaccines, it was planned that passaged virus would be tested at the highest and lowest passage levels available. One or another of these extremes might be found suitable. If necessary, further intermediate pasage levels could be developed for testing. In this approach, there was no intent to predict which, if any biological markers will correlate with virulence of virus in human beings. The identification of a successful human vaccine for one DEN type might validate biological markers of attenuation and permit improved selection of other attenuated viruses. The empiric approach to separate evaluation of multiple passage levels is based upon the precedent of modern attenuated virus vaccines; for example rubella strains that differed by only a few duck embryo passages varied markedly in human virulence (Halstead et al., 1970, JAMA 211, 911-916).
The early vaccine candidates were grown in cells. Attenuated vaccines were prepared by adaptation to growth in primary dog kidney (PDK) cells, a nonpermissive cell for dengue virus replication (Halstead 1978, Asian J. Infect. Dis. 978, 112-117). Preliminary clinical studies demonstrated that dengue virus strains could be attenuated for humans by passage in PDK cells (Eckels, 1984 , Am J Trop Med Hyg 33, 679-683; Bhamarapravati, 1987, Bull WHO 65, 189-195). PDK passage therefore provides an excellent model for those who wish to study the empirical process of selective attenuation. But, just as PDK serial passage exerts a cumulative selection process, the further passage in another cell substrate provides its own selective pressure. It is not known whether or not FRhL passge increases or decreases the virulence of virus for humans. The use of stable cell lines that must be fully characterized only one time is appealing. However, the published experience with FRhL cells suggests that these cels may reverse or destabilize biological properties acquired during serial passage in PDK (Halstead et al., 1984, Am J Trop Med Hyg 33, 654-665; Halstead et al., 1984, Am J Trop Med Hyg 33, 666-671; Halstead et al., 1984, Am J Trop Med Hyg 33, 672-678; Halstead et al., 1984, Am J Trop Med Hyg 33, 679-683; Eckels et al, 1984, Am J Trop Med Hyg 33, 679-683).
Experimental vaccines were prepared from each candidate strain of dengue virus at multiple passage levels in PDK cells; the passages empirically selected for vaccine preparation were approximately 10, 20, 30, 40, and 50. The safety and immunogenicity of various serotypes of dengue vaccine strains at one or more passage levels was then tested in volunteers. The purpose of these clinical investigations was to select candidate attenuated dengue vaccines for development as a monovalent vaccine and possible combination into a multicomponent vaccine. In this application is described the testing and selection of attenuated dengue type 2, 3, and 4 vaccines. The selection of the dengue 1 candidate vaccine has already been described in detail elsewhere (Edelman, 1994, J Infect Dis 170, 1448-1455).
The present invention satisfies the need discussed above. The present invention relates to vaccine composition comprising attenuated dengue-4 virus. The attenuated virus is provided in an amount sufficient to induce an immune response in a human host, in conjuction with a physiologically acceptable carrier and may optionally include an adjuvant to enhance the immune response of the host.
Therefore, it is one object of the present invention to provide an attenuated dengue-4 virus derived from serial passaging of a virulent dengue-4 isolate.
It is another object of the present invention to provide methods for stimulating the immune system of an individual to induce protection against dengue-4 virus. These methods comprise administering to the individual an immunologically sufficient amount of dengue-4 which has been attenuated by serial passage. The attenuated dengue-4 virus of the present invention was derived from the 341750 isolate and has been deposited on Apr. 30, 1999 under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC) of 10801 University Boulevard, Manassas, Va. 20110-2209, U.S.A., and was granted the accession number of VR-2652.
It is yet another object of the present invention to provide pure cultures of attenuated dengue-4 virus. The attenuated virus may be present in a cell culture supernatant, isolated from the culture, or partially- or completely purified. The virus may also be lyophilized, and can be combined with a variety of other components for storage or delivery to a host, as desired.